The invention relates to novel interferon-beta activity (IbA) proteins and nucleic acids. The invention further relates to the use of the IbA proteins in the treatment of interferon-beta (INF-xcex2) related disorders.
Human Interferons (IFNs) are members of a biologically potent family of cytokines. Originally, IFNs were identified as agents produced and secreted by virus-infected cells which can protect cells against further viral infections. However, in addition to this antiviral effect, IFNs can elicit many other changes in cellular behavior, including effects on cellular growth and differentiation and modulation of the immune system [e.g., see Lengyel, Annu. Rev. Biochem. 51:251-82 (1982); Gresser and Tovey, Biochim. Biophys. Acta 516(2):231-47 (1978); Gresser et al., Nature New Biol. 231(18):20-1 (1971); Dolei et al., J. Gen. Virol. 46(1):227-36 (1980); Gresser, Cell Immunol 34(2):406-15 (1977)]. By virtue of their antigenic, biological and physico-chemical properties, IFNs are classified into three groups, INF-xcex1 (leukocyte), INF-xcex2 (fibroblast) and INF-xcex3 (immune) [Stewart, J. Infect. Dis. 142(4):643 (1980)].
In humans, the IFN-xcex1 subtype encompass a multigene family of about 20 genes, encoding proteins of 166-172 amino acids that are all closely related. In contrast to this diversity, there is only one human interferon-beta (IFN-xcex2) gene, also encoding a protein of 166 amino acids. IFN-xcex2 has low homology to the IFN-xcex1 family and is an N-linked glycoprotein [Knight, Proc. Natl. Acad. Sci. U.S.A. 73(2):520-523 (1976)]. There is also only one human IFN-xcex3 gene that encodes a polypeptde of 143 amino acids that is glycosylated and forms a dimer in its native state. IFN-xcex3 shows only slight structural similarities to IFN-xcex1 or to IFN-xcex2.
All IFN-xcex1 and IFN-xcex2 (also commonly referred to as type I interferon family) appear to bind to a common high affinity cell surface receptor, a 130 kD glycoprotein that is widely distributed on different cell types and that is distinct from the one bound by IFN-xcex3. Type-I interferons are recognized by a complex containing the receptor subunits ifnar1 and ifnar2 and their associated Janus tyrosine kinases, Tyk2 and Jak1, that activate the transcription factors STAT1 and STAT2, leading to the formation of the transcription factor complex ISGF3 [interferon-stimulated gene factor 3; Li et al., Biochemie 80(8-9):703-20 (1998); Nadeau et al., J. Biol. Chem. 274(7):4045-52 (1999)]. Three distinct modes of IFN/receptor complex interaction are known: (i) INF-xcex1 with ifnar1 and ifnar2; (ii) IFN-xcex2 with ifnar1 and ifnar2; and (iii) IFN-xcex2 with ifnar2 alone [Lewerenz et al., J. Mol. Biol. 282(3):585-99(1998)]. While Lewerenz et al. suggest that INF-xcex1 and IFN-xcex2 interact with their receptors in different ways and as such may also signal differently, the events responsible for biological activity beyond receptor binding are poorly understood.
As might be predicted for such a large family of cytokines with almost ubiquitously distributed receptors, IFNs display varied physiological roles. Production of IFN-xcex1 or IFN-xcex2 is induced by infection, including viral infection or the presence of foreign cell types and antigens. It is not clear what specific molecules are responsible for induction, but double-stranded RNA and cytokines can be good inducers. There is much overlap between different cell types in both the inducers and the species of IFN that is induced. The major cell types that produce IFNs are: lymphocytes, monocytes and macrophages (for IFN-xcex1); fibroblasts and some epithelial cells and lymphoblastoid cells (for IFN-xcex2); and activated T lymphocytes (for IFN-xcex3).
In addition to the xe2x80x98classicalxe2x80x99 anti-viral activities that all IFNs elicit in their target cells, the biological consequences of IFN binding to its receptor can include inhibition of cell proliferation, induction of cell differentiation, changes in cell morphology, enhancement of histocompatibility antigen expression on many cells and stimulation of immunoglobulin-Fc receptor expression on macrophages. B lymphocytes can be induced to increase antibody production by low concentration of IFN-xcex1 or IFN-xcex2. An additional effect of IFN-xcex1 and IFN-xcex2 is activation of natural killer cells that may be responsible for the destruction of virus-infected cells or tumor cells in vivo. Overall, IFNs seem to be of great importance as part of the body""s defense against foreign organisms, foreign antigens and abnormal cell types (Clemens, in Cytokines, BIOS Scientific Publishers Limited, 1991; De Maeyer et al., in Interferons and Other Regulatory Cytokines, Wiley, New York, 1988).
INF-xcex1 and IFN-xcex2 were among the first of the cytokines to be produced by recombinant DNA technology. For example, the amino acid and nucleotide sequence of human IFN-xcex2 [Tanaguchi et al., Gene 10(1):11-15 (1980); Houghton et al., Nucleic Acids Res. 8(13):2885-94 (1980)] made it possible to produce recombinant human IFN-xcex2 in e.g., mammalian, insect, and yeast cells and in E. coli, that is free from viruses and other contaminants from human sources [e.g., Ohno and Taniguchi, Nucleic Acids Res. 10(3):967-77 (1982); Smith et al., Mol. Cell. Biol. 3(12):2156-65 (1983); Demolder et al., J. Biotechnol. 32(2):179-89 (1994); Dorin et al., U.S. Pat. No. 5,814,485 (1998); Konrad et al., U.S. Pat. No. 4,450,103 (1984)].
IFNs have been shown to have therapeutic value in conditions such as inflammatory, viral, and malignant diseases [e.g., see Desmyter et al., Lancet 2(7987):645-7 (1976); Makower and Wadler, Semin. Oncol. 26(6):663-71 (1999); Sturzebecher et al., J. Interferon Cytokine Res. 19(11):1257-64 (1999); Zein, Cytokines Cell. Mol. Ther. 4(4):229-41 (1998; Musch et al., Hepatogastroeneterology 45(24):2282-94 (1998); Wadler et al., Cancer J. Sci. Am. 4(5):331-7 (1998)]. IFN-xcex2 is a marketed drug (Betaseron, manufactured by Berlex and Avonex, manufactured by Biogen) that has been approved for use in treatment of multiple sclerosis (MS) [Arnason, Biomed Pharmacother 53(8):344-50, (1999); Comi et al., Mult. Scler. 1(6):317-20 (1996); Aappos, Lancet 353(9171):2242-3 (1999)]. IFN-xcex2 seems to reduce the number of attacks suffered by patients with relapsing and remitting MS. Betaseron, a recombinant IFN-xcex2 expressed in E. coli, consists of 165 amino acids (missing the initial methionine) and is genetically engineered so that it contains a serine at position 17, to replace a cysteine. It is a nonglycosylated form of IFN-xcex2. Avonex is a human IFN-xcex2, consisting of 166 amino acids that is produced by recombinant DNA techniques in CHO cells. This is a glycosylated form of IFN-xcex2. Also, recent studies show promising IFN efficacy in treating certain viral diseases, such as Hepatitis B or C, and cancer.
Most cytokines, including IFN-xcex2, have relatively short circulation half-lives since they are produced in vivo to act locally and transiently. To use IFN-xcex2 as an effective systemic therapeutic, one needs relatively large doses and frequent administrations. Frequent parenteral administrations are inconvenient and painful. Further, toxic side effects are associated with IFN-xcex2 administration which are so severe that some multiple sclerosis patents cannot tolerate the treatment. These side effects are probably associated with administration of a high dosage. In clinical studies it has been found that some patients produce antibodies to IFN-xcex2, which neutralize its biological activity.
Furthermore, it has been observed that dimers and oligomers of microbially produced IFN-xcex2 are formed in E. coli, rendering purification and separation of IFN-xcex2 laborious and time consuming. It also necessitates several additional steps in purification and isolation procedures such as reducing the protein during purification and reoxidizing it to restore it to its original conformation, thereby increasing the possibility of incorrect disulfide bond formation. In addition, and most likely attributable to the above-listed shortcomings, microbially produced recombinant human IFN-xcex2 has also been found to exhibit consistently low specific activity. It would be desirable, therefore, to microbially produce a biologically active IFN-xcex2 protein that has a reduced or eliminated ability to form intermolecular crosslinks or intramolecular bonds that cause the protein to adopt an undesirable structure.
To this end, variants of IFN-xcex2 sequences, applications and production procedures are known; see for example U.S. Pat. Nos. 4,450,103; 4,518,584; 4,588,585; 4,737,462; 4,738,844; 4,738,845; 4,753,795; 4,769,233; 4,793,995; 4,914,033; 4,959,314; 5,183,746; 5,376,567; 5,545,723; 5,730,969; 5,814,485; 5,869,603 and references cited therein.
Recently, the crystal structures of recombinant murine INFxcex2 [Senda et al., EMBO J. 11(9):3193-201 (1992); Mitsui et al., Pharmacol. Ther. 58(1):93-132 (1993); Senda et al., J. Mol. Biol. 253(1):187-207 (1995); Mitsui et al., J. Interferon Cytokine Res. 17(6):319-26 (1997); all of which are expressly incorporated by reference] and human INFxcex2 [Karpusas et al., Proc. Natl. Acad. Sci. U.S.A. 94(22):11813-8 (1997); Runkel et al., Pharm. Res. 15(4):641-9 (1998); Runkel et a 273(14):8003-8 (1998); Lewerenz et al., J. Mol. Biol. 282(3):585-99 (1998); all of which are expressly incorporated by reference] have been solved. Karpusas et al. determined the crystal structure of glycosylated human IFN-xcex2 at 2.2 Angstrom resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-xcex2 and human IFN-xcex12b, but displays several, distinct structural features. Like human IFN-xcex12b, INF-P contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, however, unlike human IFN-xcex12b, IFN-xcex2 dimerizes with contact surfaces from opposite sides of the molecule. Runkel et al. reported structural and functional differences between glycosylated (IFN,xcex2-1a) and non-glycosylated (IFN-xcex21b) forms of human IFN-xcex2 and suggested that the greater biological activity of INF-xcex2-1a is due to the stabilizing effect of the carbohydrate moiety.
The available crystal structure of INFxcex2 allows further protein design and the generation of more stable proteins or protein variants with an altered activity. Several groups have applied and experimentally tested systematic, quantitative methods to protein design with the goal of developing general design algorithms (Hellinga et al., J. Mol. Biol. 222: 763-785 (1991); Hurley et al., J. Mol. Biol. 224:1143-1154 (1992); Desjarlaisl et al., Protein Science 4:2006-2018 (1995); Harbury et al., Proc. Natl. Acad. Sci. U.S.A. 92:8408-8412 (1995); Klemba et al., Nat. Struc. Biol. 2:368-373 (1995); Nautiyal et al., Biochemistry 34:11645-11651 (1995); Betzo et al., Biochemistry 35:6955-6962 (1996); Dahiyat et al., Protein Science 5:895-903 (1996); Dahiyat et al., Science 278:82-87 (1997); Dahiyat et al., J. Mol. Biol. 273:789-96; Dahiyat et al., Protein Sci. 6:1333-1337 (1997); Jones, Protein Science 3:567-574 (1994); Konoi, et al., Proteins: Structure, Function and Genetics 19:244-255 (1994)). These algorithms consider the spatial positioning and steric complementarity of side chains by explicitly modeling the atoms of sequences under consideration. In particular, W098/47089, and U.S. Ser. No. 09/127,926 describe a system for protein design; both are expressly incorporated by reference.
A need still exists for proteins exhibiting both significant stability and interferon-beta activity. Accordingly, it is an object of the invention to provide interferon-beta activity (IbA) proteins, nucleic acids and antibodies for the treatment of multiple sclerosis, cancer and viral infections.
In accordance with the objects outlined above, the present invention provides non-naturally occurring interferon-beta activity (IbA) proteins (e.g. the proteins are not found in nature) comprising amino acid sequences that are less than about 97% identical to human INF-xcex2. The IbA proteins have at least one altered biological property of an INF-xcex2 protein; for example, the IbA proteins will be more stable than IFN-xcex2 and bind to cells comprising an interferon receptor complex. Thus, the invention provides IbA proteins with amino acid sequences that have at least about 3-5 amino acid substitutions as compared to the INF-xcex2 sequence shown in FIG. 1 (SEQ ID NO:1).
In a further aspect, the present invention provides non-naturally occurring IbA conformers that have three dimensional backbone structures that substantially correspond to the three dimensional backbone structure of INFxcex2. In one aspect, the three dimensional backbone structure of the IbA conformer corresponds substantially to the three dimensional backbone structure of the A-chain of INFxcex2. In another aspect, the three dimensional backbone structure of the IbA conformer corresponds substantially to the three dimensional backbone structure of the B-chain of INF-xcex2. The amino acid sequence of the IbA conformer and the amino acid sequence of INF-xcex2 are less than about 97% identical. In one aspect, at least about 90% of the non-identical amino acids are in a core region of the conformer. In other aspects, the conformer have at least about 100% of the non-identical amino acids are in a core region of the conformer.
In an additional aspect, the changes are selected from the amino acid residues at positions selected from positions 6, 13, 17, 21, 56, 59, 61, 62, 63, 66, 69, 84, 87, 91, 98, 102, 114, 118, 122, 129, 146, 150, 154, 157, 160, and 161. In a preferred aspect, the changes are selected from the amino acid residues at positions selected from positions 13, 17, 56, 59, 63, 66, 69, 84, 87, 91, 98, 114, 118, 122, 146, 157, and 161. In one aspect, the changes are selected from the amino acid residues at positions selected from positions 13, 17, 69, 84, 87, 91, 98, 118, 122, 146, 157, and 161. In another aspect, the changes are selected from the amino acid residues at positions selected from positions 13, 17, 56, 84, 87, 91, 114, 118, 122, and 161. Preferred embodiments include at least about 3-5 variations.
In a further aspect, the invention provides recombinant nucleic acids encoding the non-naturally occurring IbA proteins, expression vectors comprising the recombinant nucleic acids, and host cells comprising the recombinant nucleic acids and expression vectors.
In an additional aspect, the invention provides methods of producing the IbA proteins of the invention comprising culturing host cells comprising the recombinant nucleic acids under conditions suitable for expression of the nucleic acids. The proteins may optionally be recovered. In a further aspect, the invention provides pharmaceutical compositions comprising an IbA protein of the invention and a pharmaceutical carrier.
In an additional aspect, the invention provides methods for treating an INFxcex2 responsive condition comprising administering an IbA protein of the invention to a patient. The INFxcex2 condition includes multiple sclerosis, viral infection, or cancer.